/. exp. Biol. 155, 261-273 (1991)
261
Printed in Great Britain © The Company of Biologists Limited 1991
SEROTONIN DEPRESSES THE AFTER-HYPERPOLARIZATION
THROUGH THE INHIBITION OF THE Na + /K +
ELECTROGENIC PUMP IN T SENSORY NEURONES
OF THE LEECH
BY STEFANO CATARSI AND MARCELLO BRUNELLI*
Dipartimento di Fisiologia e Biochimica, Universitd di Pisa, Via San Zeno 31,
1-56127 Pisa, Italy
Accepted 14 August 1990
Summary
In T sensory neurones of the leech, a train of impulses elicited by intracellular
electrical stimulation leads to an after-hyperpolarization of up to 30 mV, mainly
due to the activation of the electrogenic Na + /K + -ATPase but partly to a Ca 2+ activated K + conductance. It was found that serotonin reversibly reduced the
amplitude of this after-hyperpolarization. We investigated the mechanism of
action of serotonin and found: (1) after inhibition of the Ca2+-activated K +
conductance with BaCl2 or CdCl2, serotonin was still able to reduce the afterhyperpolarization; (2) when penetration of T cells with microelectrodes leaking
sodium was preceded by serotonin perfusion of the ganglia, the normal hyperpolarization due to the activation of the electrogenic pump was converted to a
depolarization; (3) after long-lasting perfusion with K+-free saline solution (which
inhibits the Na + /K + pump), the application of CsCl caused repolarization by
reactivating the electrogenic ATPase; serotonin slowed and reduced this repolarization; (4) serotonin potentiated the depolarization of T neurones caused by the
inhibition of the N a + / K + pump following cooling of ganglia and depressed the
hyperpolarization after rewarming to room temperature. These data taken
together suggest that serotonin directly inhibits the Na + /K + electrogenic pump.
Introduction
In each segmental ganglion of the leech nervous system, three types of
mechanosensory neurones have been identified: touch (T), pressure (P) and
nociceptive (N) cells (Nicholls and Baylor, 1968). The firing discharge of these
neurones produces an after-hyperpolarization (AHP) which is involved in important physiological functions: it leads to the inhibition of the previously activated
pathway, to an elevation of the volley threshold, to a decrease of synaptic efficacy
*To whom reprint requests should be addressed.
Key words: serotonin, after-hyperpolarization, Na+/K+-ATPase, Ca2+-activated K+ conductance, leech T neurones, plasticity, Hirudo medicinalis.
262
S. CATARSI AND M .
BRUNELLI
at the axon terminals and, primarily, to a conduction block at the branching points
where small axonal processes join the main neurite (Baylor and Nicholls, 1969;
Jansen and Nicholls, 1973; Yau, 1976; Van Essen, 1973).
Existing electrophysiological research, analyzing the cellular mechanism of nonassociative learning in the swimming (Brunelli et al. 1985a, 1986; Debski and
Friesen, 1987; Nusbaum and Kristan, 1986) and shortening (Bagnoli et al. 1973;
Belardetti et al. 1982; Frank et al. 1975) behaviour patterns, triggered by a light
touch to the skin, has made it possible to demonstrate a clear effect of the
endogenous monoamine serotonin (5-HT) (Lent and Frazer, 1977; Lent et al.
1979) on the AHP of T neurones. A previous paper (Belardetti et al. 1984) has
shown that AHP amplitude can be depressed for prolonged periods either by the
activation of serotonergic Retzius cells (a pair of giant neurones located in each
segmental ganglion) or by 5-HT application. The effect is dose-dependent and is
partially blocked by the 5-HT antagonist methysergide.
In this paper we have investigated the mechanism of action of 5-HT on the AHP
of T sensory neurones. Since the AHP in these cells is partly due to the activation
of the electrogenic Na + /K + -ATPase and partly to a Ca 2+ -dependent K + conductance (gK,ca) (Baylor and Nicholls, 1969; Jansen and Nicholls, 1973; Van Essen,
1973), we performed two series of experiments: in the first, we tested the effect of
5-HT on the residual AHP after inhibiting gK,ca^ i n the second, we investigated the
effect of 5-HT on T cells in which the activity of the Na + /K + -ATPase was
modified.
The data indicate that 5-HT may act through the inhibition of the N a + / K +
electrogenic pump.
Materials and methods
Adult leeches of the species Hirudo medicinalis were purchased from a local
supplier and kept in a well-aerated aquarium at 15 °C. The animals were
anaesthetized with 10% ethanol in water and pinned, ventral side up, to the
paraffin wax floor of a plastic chamber. The ventral nerve cord was exposed by
cutting the body wall along the midline and opening the ventral sinus. A short
chain of ganglia was isolated at midbody level and pinned to the bottom of a small
recording chamber coated with Sylgard (Dow Corning).
Unless otherwise stated, the following leech saline was used: HSmmoll" 1
NaCl, 4 m m o i r 1 KC1, l . g m m o i r 1 CaCl2, lOmmolP 1 glucose, buffered to
pH7.4 with Tris-maleate.
In some experiments trains of depolarizing pulses (200 ms, 2-3 s" 1 , 25 s
duration) were injected intracellularly into T cells; the discharge frequency of each
series of trials was kept constant by adjusting the amount of current injected.
Microelectrodes filled with 3-4 moll" 1 potassium acetate with resistances ranging
from 40 to 100 MQ were used for intracellular recordings and stimulations.
In one group of experiments the temperature was lowered by circulating a
mixture of acetone and dry ice on the external surface of the recording chamber.
Serotonin and the electrogenic pump
263
The temperature of the bath was constantly monitored with an electronic thermal
probe. The shift produced by temperature variation was detected by a second
microelectrode placed in the recording chamber and algebraically subtracted from
the voltage measured by the microelectrode in the cell. The earth wire was located
far from the recording chamber, using an agar/salt bridge. Serotonin (serotonin
creatinine sulphate) (Sigma) was freshly dissolved in saline.
Only T cells with a resting potential of at least 40-50 mV, an input resistance
greater than 20 MQ and an action potential of 60-80 mV were selected. The AHP
is very sensitive to the quality of the impalement: therefore, only cells with an
AHP of at least 10-15 mV were used for the experiments.
During the recording session, the ganglia were perfused at a rate of 1.5 ml min" 1
using a peristaltic pump. All the experiments were displayed on the screen of a
storage oscilloscope and collected on a video recorder connected to a pulse code
modulator (PCM501ES) (Sony).
Results
Effect of serotonin application
We measured the effects of different concentrations of 5-HT on some electrophysiological parameters of T sensory neurones. As pointed out in a previous
paper (Belardetti et al. 1984), although a dose-dependent reduction of the AHP
occurred, we have never seen consistent changes in the shape of the action
potential (see Fig. 4A) or in the input resistance (see Fig. IB), or a modification of
the resting potential (see Fig. 1C). When SOjumolF1 5-HT was applied, the
maximal effect on AHP amplitude occurred 10 min after the removal of 5-HT by a
saline wash; with 10/imoll" 1 5-HT the maximal effect occurred 10min after 5-HT
application (Fig. 1).
Different approaches were used to investigate the mechanisms underlying 5-HT
modulation of the AHP recorded in T neurones. Responses to 5-HT were first
examined after the addition of g K;Ca blockers (BaCl2 or CdCl2), which eliminated
the component that contributes to the generation of the AHP. The effects of 5-HT
were then studied after increasing or decreasing the activity of the Na + /K + ATPase.
Effect of serotonin after application of BaCl2
In a preparation in which the AHP had been generated by trains of action
potentials elicited by intracellular depolarizing pulses, CaCl2 was completely
replaced by BaCl2 (1.8mmol I"1) to block gK,ca from the intracellular side of the
Ca 2+ channel (Hagiwara, 1981; Meech, 1978; Paupardin-Tritsch et al. 1981). A
5 min application of BaCl2 produced a reversible reduction of AHP amplitude to
60% of the initial response (taken as 100%). Prolonged applications (up to
30 min) did not reduce the AHP to a greater degree (Fig. 2B). During the 5 min
BaCl2 application the spike shape changed dramatically: the repolarization slowed
and the undershoot disappeared (see Fig. 4B). After the initial 5 min of BaCl2
264
S. CATARSI AND M. BRUNELLI
lOOn A
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a.
X 40200J
E
11010090-
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-4=4=
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V=5
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90 J
Saline
5-HT
0
Saline
10
15
20
25
30
35
40
Time (min)
Fig. 1. (A) Trains of depolarizing pulses were injected intracellularly into T neurones.
The preparation was perfused for 10 min with 50 ^mol 1~' (•) or 10 ^mol 1~L 5-HT (O).
The after-hyperpolarization (AHP) amplitude of each cell (mean+s.E.M.) refers to the
initial AHP amplitude, taken as 100% (also in Figs 2 and 3). A prolonged and dosedependent reduction of the AHP amplitude was observed. (B) Input resistance (Rm)
during the experiment illustrated in A. (C) Resting potential (Vm) during the
experiment illustrated in A. N indicates the number of experiments performed (in all
the figures).
application, perfusion with the blocking agent was continued and 50 or lO/imolF 1
5-HT was applied for 10 min. A further clear-cut reduction of AHP amplitude (to
25% of the initial response with 50//moll" 1 5-HT or 40% with 10//moll" 1 5-HT)
was observed (Fig. 2), which agreed with the kinetics shown by 5-HT alone
(Fig. 1A). The effect of the monoamine reversed after a 15 min wash in saline with
BaCl2, and an additional 15 min of perfusion with normal physiological solution
reversed the effect of BaCl2, restoring, almost completely, the AHP amplitude.
The effects are illustrated and summarized in Fig. 2B.
Effect of serotonin after application of CdCl2
A similar experiment was performed using 0.1 mmol P 1 CdCl2 in normal saline
Serotonin and the electrogenic pump
265
BaCl2 (l.SmmoirO+S-HT (5x10^moll" 1 )
10 mV
BaCl 2 5min
Control
BaCl 2 +5-HT
10 min
t
t Wash in saline
| Wash in BaCl 2
15 min
|
10 s
15 min
B
N=U
100-
80-
U 60en
3 40H
20-
0-
Saline
BaCl 2
0
BaCl 2 +5-HT
10
Saline
BaCl 2
15
20
25
30
35
40
45
Time (min)
Fig. 2. (A) A train of depolarizing pulses was injected intracellularly into a T cell. The
preparation was then perfused for 5 min with saline in which CaCl2 was completely
replaced with BaCb (1.8mmoll~l); another depolarizing train was then delivered. A
reduction of the AHP amplitude was observed. Perfusion with BaCl2 was maintained
for up to 30 min and 50/zmoll~' 5-HT was applied for 10 min; we observed a further
clear-cut reduction of the AHP. The effect of the monoamine reversed after 15 min of
perfusion in saline with BaC^; an additional 15 min perfusion with normal physiological solution reversed the effect of BaCl2. (B) Graph showing the mean+s.E.M. of the
AHP amplitudes during ( • ) prolonged application of BaCl2, (O) application of BaCl2
and lOjjmoir 1 5-HT, and ( • ) application of BaCl2 and SO^moll"1 5-HT.
to block gK,Ca from the extracellular side of the Ca 2+ channel (Hagiwara, 1981;
Madison and Nicoll, 1982; Meech, 1978) (Fig. 3). The same results were obtained
as in the previous experiment. In this case the trace was less noisy with no
spontaneous spikes after the testing train: this is probably attributable to the fact
266
S. CATARSI AND M. BRUNELLI
CdCl2 (0.1mmoll"')+5-HT
10 mV
CdCl 2 5min
Control
I Wash in CdCl2
15min
CdCl 2 +5-HT
10 min
I Wash in saline
15 min
10s
lOO-i
80N=9
§• 60'
ft.
X
< 40'
"o
S?
'
20-
Saline
CdCl2
0
CdCl 2 +5-HT
10
CdCl2
15
20
Saline
25
30
35
40
45
Time (min)
Fig. 3. (A) 0.1 mmol I 1 CdCl2 in normal physiological solution was applied for 5 min,
producing a reduction of AHP amplitude. Continued perfusion with this blocking
agent plus 50/anolF 1 5-HT for 10 min gave a further reversible reduction in AHP
amplitude. (B) Graph showing the mean+s.E.M. of the AHP amplitudes during:
(•) prolonged application of CdCl2, (O) application of CdCl2 and lO/imoll"1 5-HT,
and (•) application of CdCl2 and 50fimo\ I"1 5-HT.
that, in the experiment with BaCl2, Ca 2+ -free solution was used and consequently
cellular excitability increased. A 5 min application of CdCl2 induced a decrease of
AHP amplitude to 60% of the initial response, with no further reduction when
application was continued for up to 30 min (Fig. 3B). A 5 min application of CdCl2
caused an increase in spike duration and a disappearance of the undershoot
(Fig. 4C). A 10 min incubation with 5-HT led to a further depression of the AHP
amplitude, to approximately 30 % of the initial response with 50 jumol I" 1 5-HT or
approximately 45% with lO/imoir 1 5-HT (Fig. 3). The effect was partially
restored after washing out the 5-HT (Fig. 3B).
Serotonin and the electrogenic pump
267
20 mV
CdCl2
Control
Fig. 4. (A) Action potential shape before and after lOmin of perfusion with
50/mioir 1 5-HT. (B) Changes in the action potential shape after 5min of BaCl2
application. (C) Changes in the action potential shape after 5 min of CdCl2 application.
Effect of serotonin after injection of Na+
When T neurones were impaled with a 3 mol I" 1 sodium acetate microelectrode
a clear hyperpolarization (about 15 %) could be observed (Fig. 5A). It is assumed
that this effect is due to the activation of the Na + /K + electrogenic pump following
Na + leakage from the microelectrode into the cell (Jansen and Nicholls, 1973).
When potassium acetate microelectrodes were used, no variation of membrane
voltage (Vm) could be detected (Fig. 5B).
In some experiments we perfused the ganglia with SO^molP 1 5-HT for 10 min
before penetrating T cells with sodium acetate electrodes and we obtained a
depolarization of membrane voltage of about 15 % (Fig. 5C). This effect may be
explained by an inhibitory action of 5-HT on the Na + /K + -ATPase so that Na +
leaking into T cells cannot be pumped out and a depolarization results. Fig. 5D
clearly illustrates the comparative membrane voltages of T neurones after
penetration with 3 mol 1 1 potassium acetate or 3 mol 1 1 sodium acetate with or
without the application of 5-HT.
Effect of serotonin after application of CsCl
In experimental preparations in which the ganglia were perfused with K+-free
saline, we obtained a biphasic variation of membrane voltage. An early hyperpolarization phase, probably due to a greater K + outflow caused by the rise of the
chemical gradient, was followed by a delayed depolarization phase which was
sensitive to ouabain and, therefore, mainly due to the inactivation of the
electrogenic pump, which is known to be sensitive to the external K + concentration (Fig. 6A). These results agree with those found in P and N cells by Schlue
and Deitmer (1984).
If, at the end of the depolarization phase, we perfused the ganglia with K + -free
268
S. CATARSI AND M. BRUNELLI
-42mV •
-44 mV10 mV
- 4 4 mV'
N=19
N=U
N=4O
0
1
2
3
4
Time (min)
5
Fig. 5. (A) Membrane potential (Vm) of a T neurone after penetration with a 3 mol P 1
sodium acetate microelectrode. A hyperpolarization due to the activation of the
electrogenic pump can be observed. (B) Vm of a T neurone after penetration with a
3 m o l l " 1 potassium acetate microelectrode. No variations of Vm were observed.
(C) Vm of a T neurone after penetration with a 3 mol I" 1 sodium acetate microelectrode in a preparation perfused for lOmin with SO^molF 1 5-HT before penetration.
A clear-cut depolarization from the initial Vm was noted. (D) Mean±s.E.M. of Vm:
(O) 3 moll" 1 sodium acetate microelectrode; ( A ) S m o i r 1 potassium acetate microelectrode; ( • ) 3 mol I" 1 sodium acetate microelectrode plus a 10 min exposure to
5 0 / m i o i r 1 5-HT. In all the cases Vm values of each cell are referred to the initial
resting potential, taken as 100%. This also applies to Figs 6 and 7.
saline containing 4 mmol I" 1 Cs + , a Na + /K + -ATPase activator (Skou, 1960,1965),
we observed a membrane repolarization (Fig. 6C). Alternatively, if SO^moll" 1
5-HT was applied 10 min before CsCl, the repolarization was greatly reduced (by
approximately 30%) and delayed (Fig. 6B).
Effect of serotonin after cooling the ganglia
The Vm of T neurones was monitored during the reversible cooling of the
preparation from 18 to 10°C. During cooling a depolarization of about 15 % was
Serotonin and the electrogenic pump
60'
269
[K + ]=0mmoir'
yv=5
A'=5
80-
E
100-
yv=5
«4-i
o
3? 120
140
160
10
15
20
Time (min)
25
30
35
Fig. 6. Mean±s.E.M. of Vm for T neurones (N=5) perfused with K + -free saline for
about 25 min (NaCl was raised to 119 mmol I" 1 to maintain iso-osmolarity). Two phases
are evident: a hyperpolarization followed by a depolarization. (D) Prolonged
application of K + -free solution (A). (O) After depolarization, five cells were perfused
with saline in which 4 mmol I" 1 KC1 was completely replaced with 4 mmol I" 1 CsCl. A
repolarization occurred (C). ( • ) After depolarization, five cells were treated for
10 min with 5 0 / m i o i r 1 5-HT and then perfused with saline containing CsCl.
Repolarization was reduced (B).
obtained (Fig. 7). When the temperature was restored to 18°C a repolarization
was observed; this was sensitive to ouabain and strophantidin and often rose to
105-110 % of the initial Vm. This effect is due to the inhibition and reactivation of
the Na + /K + -ATPase (Baylor and Nicholls, 1969; Kuba and Koketsu, 1979).
The same procedure was adopted after a 10 min application of 50 jiimol I" 1 5-HT,
producing a larger depolarization phase of about 25-30% and a smaller
repolarization phase reaching 90% of the initial voltage (Fig. 7).
The effect of serotonin after ouabain treatment
The Na /K -ATPase was blocked with 2 x l O ~ 4 m o i r 1 ouabain or strophantidin in order to study the effect of 5-HT on the residual AHP; however, the
remaining AHP was often very small (Baylor and Nicholls, 1969; Jansen and
Nicholls, 1973) and it was difficult to maintain a constant discharge frequency,
probably because of an excess of Na + in the T cell that cannot be pumped out. In a
few cases in which a residual AHP could be detected, 5-HT was unable to provoke
a further reduction of the AHP amplitude (data not shown).
+
+
Discussion
In a previous paper (Belardetti et al. 1984) it was shown that 5-HT perfusion or
270
S. CATARSI AND M. BRUNELLI
70-,
90-
A'=6
100110J
Temperature (°C)
Fig. 7. (O) Mean+s.E.M. of Vm in T neurones after cooling to 10°C and rewarming to
room temperature (18°C). During cooling a depolarization took place, whereas during
rewarming repolarization was observed. This is due to the inhibition and reactivation
of the electrogenic pump. ( • ) lOmin before cooling, the ganglia were perfused with
50/imoll" 1 5-HT. The application lasted lOmin after the cooling process had started.
This produced a greater depolarization phase and a slower arid smaller repolarization
one. The entire cooling and rewarming procedure lasted 15-20min.
the electrical stimulation of the serotonergic Retzius cells produced a long-lasting
clear-cut reduction of AHP amplitude in T sensory neurones. This effect
depended on the concentration of the monoamine, was inhibited by the antagonist
methysergide and was not blocked by bathing the ganglion with high-Mg2+ saline.
In agreement with the observations of Belardetti et al. (1984), we have never seen
consistent changes of T cell resting potential, input resistance or action potential
shape with 5-HT perfusion (Figs 1, 4A).
From these preliminary observations, it was difficult to ascribe the mechanism
of action of serotonin to an inhibition of gK,Ca> which is known to make a partial
contribution to the AHP (Baylor and Nicholls, 1969; Jansen and Nicholls, 1973); in
fact, no increase of input resistance was observed. A simple short-circuit of the
currents generating the AHP probably also fails to explain the action of 5-HT; a
70-75% decrease of the AHP amplitude, obtained with 50/mioir 1 5-HT
perfusion for 10 min, should result from an identical reduction of the input
resistance. A possible shunting action of 5-HT is also unlikely because it would
affect the AHPs identically, irrespective of their amplitudes, whereas large AHPs
are more depressed than the small ones (Belardetti et al. 1984).
To clarify the mechanism of action of 5-HT on T neurones, we performed two
series of experiments. In the first series, we blocked gK,ca with BaCl2 or CdCl2 and
demonstrated that 5-HT was still able to reduce AHP, even after gK.ca inhibition.
We also observed that Ba 2+ or Cd 2+ application caused an unexpectedly large
reduction (about 40 %) of the AHP amplitude, which is known to depend greatly
Serotonin and the electrogenic pump
271
on the activation of the electrogenic pump in T neurones. This may be caused by
the reduction of input resistance that accompanies BaCl2 or CdCl2 application
(about 16% and 11 %, respectively), probably due to an increase of membrane
permeability following variation of Ca 2+ concentration. Moreover, an interaction
between gK,ca a n d electrogenic pump activity cannot be ruled out. An involvement of 5-HT in gK,ca is made unlikely by the finding that, in experiments in which
it was possible to get a residual AHP following ouabain or strophantidin
treatment, 5-HT was ineffective in provoking a further reduction of the AHP
amplitude.
We also tried to study the effect of 5-HT by holding T cells at a membrane
potential close to the K + reversal potential, but the long projections of the neurite
do not allow good space-clamping even with double-electrode voltage-clamp
techniques (Jansen and Nicholls, 1973; Stewart et al. 1989).
In the second series of experiments, it has been possible to collect evidence for a
direct inhibition of the Na + /K + -ATPase by 5-HT. Injection of Na + into T
neurones after perfusion with 5-HT led to a depolarization instead of the normal,
ouabain-sensitive hyperpolarization. This effect may be explained by an inhibition
of the electrogenic pump by 5-HT and by leakage of positive ions into the cell.
These ions cannot then be pumped out.
Moreover, 5-HT caused a reduction of the repolarization normally obtained
with the N a + / K + pump activator Cs + after K+-free perfusion. Cs + has a
permeability of less than 8 % for the K + channel (taking the permeability of K + for
its channel as 100%) (Stryer, 1982) and, therefore, its effect on the N a + / K +
electrogenic pump is not masked by the current through the K + channel, as
happens when K + itself is used to reactivate the pump.
In addition, 5-HT increased the depolarization phase, during cooling of the
ganglia and produced a considerable reduction in speed of repolarization when the
temperature was returned to 18°C and, consequently, the electrogenic pump
should have been reactivated. The inhibitory action of 5-HT on the electrogenic
pump is a surprising result since, although examples of positive modulation of the
Na + /K + -ATPase caused by neurotransmitters have been described (Kuba and
Koketsu, 1979; Phillis and Wu, 1981; Thomas, 1972), inhibitory effects have rarely
been found and have been investigated only with biochemical methods (Lingham
and Sen, 1983; Mourek, 1988).
A further observation is that 5-HT has a reduced effect on the AHP amplitude
of P sensory cells and no effect at all on that of N neurones (Brunelli et al. 19856),
the AHP of which is more dependent on gj<,ca (Baylor and Nicholls, 1969; Jansen
and Nicholls, 1973; Van Essen, 1973).
A possible effect of 5-HT on the Na + /K + -ATPase might be to change the
transport coupling ratio, making the pump work with a less electrogenic
mechanism. Through the inhibition of the electrogenic pump, 5-HT may have a
role in removing the conduction blocks caused by the AHP. This potentiating
effect may, at least in part, explain the behavioural sensitization and dishabituation induced by 5-HT and observed in experiments on the induction of swimming
272
S. CATARSI A N D M . BRUNELLI
(Brunelli et al. 1985a, 1986; Debski and Friesen, 1987; Nusbaum and Kristan,
1986) and on the 'fast conducting system' (Belardetti et al. 1982; Frank et al. 1975).
It- will be interesting to investigate whether a similar inhibitory action of the
electrogenic pump can be found in other leech neurones, in other nervous systems
or in non-excitable tissues.
This work was supported by the Ministero della Pubblica Istruzione: 40-60%,
1987-1988. Additional funding was provided by Regione Toscana, Italy, grant
number 3342, 1987.
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